title: The titanium binding protein of Rhodococcus ruber GIN1 (NCIMB 40340) is a cell-surface homolog of the cytosolic enzyme dihydrolipoamide dehydrogenase.
abstract: Rhodococcus ruber GIN1 (formally Rh. strain GIN1) was previously isolated on the basis of its strong adherence to coal fly ash (CFA) and titanium dioxide particles from CFA sedimentation ponds of an electrical power plant in Israel. The interaction of the bacterium with oxides has been shown to be mediated by a cell surface protein designated TiBP (titanium binding protein) involving primarily strong, non-electrostatic forces. In this work, we set forward to identify this unique exocellular protein. Sequence analysis of the purified protein by mass spectrometry (LC/MS/MS) following trypsinization revealed 11 peptides. All of them showed >90% amino acid residues identity with sequences of one of the orthologs (dldh1) of the cytosolic enzyme dihydrolipoamide dehydrogenase (DLDH), based on the genome sequence of Rhodococcus strain RHA1. This genome was selected as a reference since currently it is the only sequenced Rhodococcal genome. Altogether, these peptides covered over 25% of the 52 kDa protein molecule. N- and C-termini primers were prepared and used to sequence the paralog gene from Rh. ruber GIN1 after polymerase chain reaction (PCR) amplification. All 11 peptides showed 100% identity with the sequence of this gene. The homology of TiBP with the supposedly cytosolic DLDH raised the question of whether the exocellular TiBP possesses DLDH activity. Indeed, intact late logarithmic phase Rh. ruber GIN1 cells, previously shown to express TiBP, were found to possess such activity, while very low activity was associated with stationary phase cells which possess diminished TiBP expression on their surface. Further evidence for the exocellular location of TiBP/DLDH was achieved using specific anti-TiBP polyclonal antibodies by whole cell and protein enzyme-linked immunosorbent assay (ELISA), showing high reactivity of the logarithmic phase cell surface and substantially lower reactivity with the stationary phase cells. As expected, logarithmic phase spheroplasts were not recognized by these antibodies. Similar results were obtained by fluorescence and scanning electron microscopy. Our postulation that DLDH is located on the surface of Rh. ruber GIN1, serving as a TiO2 binding protein, is in accordance with literary evidence on DLDH in other organisms, Bacteria, Archea, and Eukaryots that suggests it is associated with the outer membranes or cell surfaces. As an exocellular protein DLDH assumes various tasks which are not related to its classical role as a 2-oxoacid dehydrogenase, including serving as an adhesion/binding protein in certain bacteria.
authors.affiliation: Department of Molecular Microbiology and Biotechnology, The George Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv, Israel.
authors.forename: Ari
authors.initials: A
authors.lastname: Siegmann
authors.forename: Avital
authors.initials: A
authors.lastname: Komarska
authors.forename: Yifaat
authors.initials: Y
authors.lastname: Betzalel
authors.forename: Irene
authors.initials: I
authors.lastname: Brudo
authors.forename: Sadanari
authors.initials: S
authors.lastname: Jindou
authors.forename: Gil
authors.initials: G
authors.lastname: Mor
authors.forename: Gideon
authors.initials: G
authors.lastname: Fleminger
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D003600:Cytosol / Q000201:enzymology*; D008058:Dihydrolipoamide Dehydrogenase / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D007074:Immunoglobulin G / Q000276:immunology; D008969:Molecular Sequence Data; D011817:Rabbits; D012240:Rhodococcus / Q000737:chemistry / Q000201:enzymology* / Q000254:growth & development; D017386:Sequence Homology, Amino Acid; D013104:Spheroplasts / Q000378:metabolism; D014025:Titanium / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D007074:Immunoglobulin G; C009495:titanium dioxide; D014025:Titanium; D008058:Dihydrolipoamide Dehydrogenase